Antibacterial and antifungal activities of new acylated derivatives of epigallocatechin gallate

Abstract

(-)-Epigallocatechin-3-O-gallate (EGCG) has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori et al. (2008) found that addition of long acyl chains (C16-18) to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal MICs of the derivatives were also two to fourfold lower than those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate and palmitate (C16) for 17 Staphylococcus aureus strains were 4-32 μg/ml, although MIC of EGCG for these 17 strains was ≥128 μg/ml. C16 demonstrated rapid bactericidal activity against methicillin-resistant S. aureus (MRSA) ATCC43300 at ≥16 μg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane-permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported in Escherichia coli using the efflux pump AcrAB-TolC. The tolC deletion mutant exhibited higher sensitivity to EGCG and the derivatives than wild-type. Addition of long alkyl chains to EGCG significantly enhanced its activities against several bacteria and fungi, particularly against S. aureus including MRSA. C16 might potentially become under specified circumstances an alternative or supplement to antibiotics and disinfectants in the future.

Keywords: bactericidal activity Staphylococcus aureus; epigallocatechin gallate; fatty acid esters.

Figure 1

Lipase-catalyzed synthesis of EGCG fatty acid esters. One or two of R^1–4 are substituted with fatty acids.

Figure 2

Acyl chain length of EGCG derivatives and their antistaphylococcal activities against MSSA ATCC25923 (A) and MRSA ATCC43300 (B). Mean ∓ SD of each four wells optical density (600 nm) after 18 h incubation are shown.

Figure 3

Killing activity of C16 against MRSA ATCC43300 (A), E. coli MG1655 (B), and P. aeruginosa PAO1 (C). Cells were incubated with C16 in Muller–Hinton broth at 37°C on a shaker. Residual cells after 2, 4, and 6 h incubation were counted as colonies grown on the Mueller–Hinton agar plates. Mean ∓ SD of the residual CFUs calculated from two to four plates are shown.

Figure 4

Combinatorial effects of C16 with IPM (A,B) or AMP (C,D) against MRSA strains. Optical densities (600 nm) after 18 h incubation were determined by the checkerboard method.

Figure 5

Combinatorial effects of EGCG with IPM (A,B) or AMP (C,D) against MRSA strains. Optical densities (600 nm) after 18 h incubation were determined by the checkerboard method.

Figure 6

Membrane damage caused by C16 determined by SYTOX Green uptake assay. Fluorescence was determined (Ex/Em: 504/523 nm) after 10 min incubation with SYTOX Green in a black 384-well plate using a microplate reader after incubation with the antimicrobial compound for 15 min. Fluorescence is shown as a ratio to that of control. Data represent Mean ∓ SD (n = 3). *A polycyclic antibacterial peptide.